The proteasome is one of the largest protein complexes to be found in cells. This multi-subunit ATP-dependent protease controls numerous cellular processes by degrading carefully selected targets. Proteasome function is essential for all eukaryotic cells. Defects in the proteasomal degradation pathway are associated with various diseases, including cancers, neurodegenerative disorders, developmental disorders, immune and inflammatory disorders, and muscle wasting. How proteins destined for degradation are recognized by the proteasome and then degraded is only partially understood. Precise knowledge of proteasome subunit architecture, their interactions with each other, and associations with transient auxiliary factors is critical if we are to understand its mode of action. Such information would also serve as the basis for ongoing drug design efforts. Targeting of polyubiquitinated substrates to the proteasome is mediated by several shuttle proteins (e.g., Dsk2, Rad23, Ddi1), all of which contain a ubiquitin-like (UBL) domain that interacts directly with several proteasomal subunits. Of these subunits, the primary UBL-receptor is apparently Rpn1. The goal of the proposed research is to understand how the proteasome differentiates between ubiquitin and ubiquitin-like signals. We will do so by characterizing, at the biochemical and structural levels, the nature and the strength of interactions involved in recognition of the UBL signals by the proteasome. We will map UBL-binding regions on Rpn1, determine the three-dimensional structure of specialized UBL-receptor regions at an atomic level resolution and characterize the nature and the strength of these interactions both structurally and biochemically. We will compare recognition of UBLs to understand how they differ among themselves, and how as a group they pose a distinct signal from ubiquitin which binds to designated receptors. We will address the ability of the proteasome to differentiate between ubiquitin and ubiquitin-like proteins, and the apparent redundancy in signaling by polyubiquitin and UBLs. All structural predictions and pairs-wise interactions will be verified through biological manipulations and proteomic screens. We will complement the in vitro characterization with verification of interactions in cells, test the outcome of mutants designed to abolish or strengthen pair-wise interactions that we identify, alter hierarchy of proteasome targeting, and look at the broad proteomic role of each shuttle, UBL signal, or receptor individually. This project relies on a variety of experimental approaches (biochemical, biophysical, structural (NMR), and proteomic) to reveal the structure-function relationship of the proteasome with its substrates, targets, and signals. These studies will provide a detailed picture of molecular events involved in the recognition of the UBL signals by the proteasome, triggering subsequent events at the proteasome leading to substrate degradation.